Episomal expression of sense and antisense insulin-like growth factor (IGF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I

Pomila Singh, Bosong Dai, Bharati Dhruva, Steven G. Widen

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 107 cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 ± 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells. In the present study, we have, for the first time, demonstrated a potent inhibitory role of endogenous IGFBP-4 for regulating the mitogenic response of the cells to both endogenous and exogenous IGFs. An important observation was that the inhibitory effects of endogenous IGFBP-4 could not be titrated out by the addition of an excess of IGF-I or insulin; the addition of IGFBP-4 antibody, on the other hand, could effectively remove this block. The latter findings suggest that IGFBP-4 may be inhibiting the mitogenic effects of IGF-I by other possible IGF-independent mechanisms.

Original languageEnglish (US)
Pages (from-to)6563-6570
Number of pages8
JournalCancer Research
Volume54
Issue number24
StatePublished - Dec 15 1994

Fingerprint

Insulin-Like Growth Factor Binding Protein 4
Insulin-Like Growth Factor I
Colonic Neoplasms
Complementary DNA
Cell Line
HT29 Cells
Conditioned Culture Medium
Growth
Insulin-Like Growth Factor II
Antibodies
Somatomedins
Antisense DNA

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

@article{fb5c4ef804914bc3ac144c2df0543581,
title = "Episomal expression of sense and antisense insulin-like growth factor (IGF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I",
abstract = "HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 107 cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 ± 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells. In the present study, we have, for the first time, demonstrated a potent inhibitory role of endogenous IGFBP-4 for regulating the mitogenic response of the cells to both endogenous and exogenous IGFs. An important observation was that the inhibitory effects of endogenous IGFBP-4 could not be titrated out by the addition of an excess of IGF-I or insulin; the addition of IGFBP-4 antibody, on the other hand, could effectively remove this block. The latter findings suggest that IGFBP-4 may be inhibiting the mitogenic effects of IGF-I by other possible IGF-independent mechanisms.",
author = "Pomila Singh and Bosong Dai and Bharati Dhruva and Widen, {Steven G.}",
year = "1994",
month = "12",
day = "15",
language = "English (US)",
volume = "54",
pages = "6563--6570",
journal = "Journal of Cancer Research",
issn = "0008-5472",
publisher = "American Association for Cancer Research Inc.",
number = "24",

}

TY - JOUR

T1 - Episomal expression of sense and antisense insulin-like growth factor (IGF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I

AU - Singh, Pomila

AU - Dai, Bosong

AU - Dhruva, Bharati

AU - Widen, Steven G.

PY - 1994/12/15

Y1 - 1994/12/15

N2 - HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 107 cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 ± 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells. In the present study, we have, for the first time, demonstrated a potent inhibitory role of endogenous IGFBP-4 for regulating the mitogenic response of the cells to both endogenous and exogenous IGFs. An important observation was that the inhibitory effects of endogenous IGFBP-4 could not be titrated out by the addition of an excess of IGF-I or insulin; the addition of IGFBP-4 antibody, on the other hand, could effectively remove this block. The latter findings suggest that IGFBP-4 may be inhibiting the mitogenic effects of IGF-I by other possible IGF-independent mechanisms.

AB - HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 107 cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 ± 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells. In the present study, we have, for the first time, demonstrated a potent inhibitory role of endogenous IGFBP-4 for regulating the mitogenic response of the cells to both endogenous and exogenous IGFs. An important observation was that the inhibitory effects of endogenous IGFBP-4 could not be titrated out by the addition of an excess of IGF-I or insulin; the addition of IGFBP-4 antibody, on the other hand, could effectively remove this block. The latter findings suggest that IGFBP-4 may be inhibiting the mitogenic effects of IGF-I by other possible IGF-independent mechanisms.

UR - http://www.scopus.com/inward/record.url?scp=0028659793&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028659793&partnerID=8YFLogxK

M3 - Article

C2 - 7527300

AN - SCOPUS:0028659793

VL - 54

SP - 6563

EP - 6570

JO - Journal of Cancer Research

JF - Journal of Cancer Research

SN - 0008-5472

IS - 24

ER -