1. Volume 123, Issue 4, Pages 1191-1204 (October 2002)
Effects of genetic blockade of the insulin-like growth factor receptor in human colon cancer cell lines 
Yasushi Adachi, Choon–Taek Lee, Keith Coffee, Noboru Yamagata, Joyce E. Ohm, Kyung–Ho Park, Mikhail M. Dikov, Sorena R. Nadaf, Carlos L. Arteaga, David P. Carbone 
Gastroenterology 
Volume 123, Issue 4, Pages (October 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Stable transfection of tetracycline-repressible 482/stop of IGF-Ir cDNA into human colon cancer HT29 cells. (A) Confirmation by PCR using primers specific for the presence and expression of the construct. The truncated IGF-Ir mRNA was not expressed by RT-PCR in the presence of tetracycline (lane 1) but was detectable in the HT29dn transfectant in the absence of tetracycline (lane 2). PCR of genomic DNA from HT29dn (lane 4) showed a band corresponding to that seen with the expression vector plasmid (lane 5) and not in the parental cell DNA (lane 3) or the empty vector (lane 6). cDNA, cDNA generated after deoxyribonuclease; gDNA, genomic DNA; pDNA, plasmid DNA; tetracycline, tetracycline used in the culture of the cells. (B) HT29dn cells were cultured in conditioned media with or without tetracycline and washed with serum-free medium twice before culturing in serum-free media with or without tetracycline for 6 hours or 24 hours to collect produced proteins. The same amount of cultured medium was collected for each condition, and both were concentrated 50 times and loaded on a 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis gel. Western blotting showed that HT29dn secreted a short IGF-Ir (arrow) into the medium in the absence of tetracycline.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
(A) Low-power (upper panels) and high-power (lower panels) views show that HT29dn cells were able to form many large colonies in soft agar containing tetracycline but not in its absence. (B) Summary of quadruplicate experiments of soft agar colony-forming assays, showing the effect of the soluble form of IGF-Ir on suppression of this in vitro surrogate for tumorigenicity. The colony numbers of HT29dn in complete medium without tetracycline were significantly reduced (*P = ) compared with that of HT29dn cultured with tetracycline.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
IGF-Ir/tf enhances apoptotic responses to serum starvation and heat shock. Annexin V expression shows apoptosis induction by 24 hours of serum starvation or heat shock (42°C for 30 minutes). The number of cells positive for annexin V and negative for propidium iodide, detected by fluorescence-activated cell sorter, represents the apoptotic fraction. Summarized results of 3 independent experiments show that tetracycline withdrawal increased apoptosis 2–3 times induced by heat shock alone, starvation alone, and heat shock and starvation together compared with cells with these stressors cultured in tetracycline-containing medium. Induced expression of the soluble form of IGF-Ir caused a significant increase in the apoptotic fraction after serum starvation or starvation plus heat shock compared with the cells without stressors. Bars show means and SE (*P < 0.05 for tetracycline +/− comparison).
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Chemotherapy-induced apoptosis as measured by annexin V expression was increased by soluble IGF-Ir/tf induction in HT29dn cells. (A) In the presence of the soluble form of IGF-Ir, apoptosis was more effectively induced without chemotherapy drugs, after treatment with 260 μmol/L 5-FU, and after treatment with 10 μmol/L cisplatin (CDDP) compared with HT29dn in which truncated receptor expression was suppressed by tetracycline (*P < 0.01). Summarized results of 3 independent experiments are shown. (B) Annexin V assay (after 24 hours) showed that the presence of the soluble form of IGF-Ir increased the fraction of cells undergoing apoptosis and increased the apoptosis in response to 5-FU. The summarized results of 3 independent experiments are shown (*P < 0.05). (C) Apoptosis of HT29dn cells cultured with tetracycline was increased by treatment with wortmannin, a PI-3 kinase inhibitor without 5-FU, or with 5-FU but not by PD98059, a MEK1 inhibitor. There was a significant difference in 5-FU–induced apoptosis between tetracycline-suppressed cells treated with PD98059 and derepressed-expressing cells treated without inhibitor, but 5-FU–induced apoptosis of tetracycline-suppressed cells with wortmannin was not different from that of derepressed cells without inhibitor (*P < 0.01). (D) The increased apoptosis in HT29dn without tetracycline was reduced to baseline by infection with adenovirus vector encoding myristoylated active Akt (AxCAMyr-Akt), *P < 0.01 compared with soluble IGF-Ir secreting cells treated with 5-FU. These data suggest that the main antiapoptotic signal of IGF-Ir in these cells is via Akt-1.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
(A) Equal aliquots of whole cell lysates were immunoprecipitated with anti–IRS-1 and analyzed by Western blotting and then detected by anti–phospho-tyrosine (upper panel) and anti–IRS-1 (lower panel). IGF-II phosphorylated IRS-1 in HT29dn cultured with tetracycline, however, not in the presence of the truncated form of IGF-Ir (without tetracycline). (B) Western blotting showed that phosphorylated Akt and phosphorylated MAPK were seen after stimulation with 10 ng/mL IGF-II in HT29 cells and HT29dn cells with tetracycline. Akt phosphorylation was reduced in the presence of truncated IGF-Ir (after tetracycline withdrawal), but there were no differences in MAPK phosphorylation. (C) The truncated form of IGF-Ir was able to block IGFs inducing Akt kinase activation. Time-dependent phosphorylation of the Akt kinase substrate GSK-3β was blocked by the withdrawal of tetracycline in HT29dn cells. Although the baseline of Akt kinase activity was higher in the series stimulated with IGF-I than that with IGF-II, Akt kinase activity was not up-regulated by IGFs after both 5- and 20-minute stimulations in the presence of the short form of IGF-Ir.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Demonstration of the bystander effect of IGF-Ir/tf in a 2-chamber system. Parental HT29 cells were cultured in the lower chamber and HT29dn cells were seeded in the upper chamber separated by a membrane too small to allow cell transmigration but large enough to pass soluble IGF-Ir/482st. After HT29 cells were stimulated with IGFs with or without tetracycline, whole cell proteins were extracted from the parental HT29 cells and Western blotting was performed. Akt phosphorylation in parental HT29 cells was blocked in medium without tetracycline, indicating that IGF-Ir/tf has a bystander effect mediated by a diffusible factor. The phospho-Akt/Akt ratios were calculated by densitometry as follows: 0.1 in wild-type IGF-Ir without stimulation, 0.1 in the truncated form of IGF-Ir without stimulation, 0.8 in tetracycline (+) with IGF-I stimulation, 0.5 in tetracycline (−) with IGF-I, 0.7 in tetracycline (+) with IGF-II stimulation, and 0.3 in tetracycline (−) with IGF-II.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Suppression of HT29dn xenograft growth after induction. (A) HT29dn cells and HT29 parental cells were inoculated subcutaneously into nude mice. Half of the HT29dn-bearing mice were fed water with tetracycline, and the others were fed regular water. There were significant differences in the tumor sizes among these groups compared with HT29dn tumors in mice fed tetracycline-free water. *P < (B) Suppression of established HT29dn tumors after induction. Mice were fed tetracycline-containing water at the time of tumor cell inoculation. After forming visible tumors, half of the mice were switched to drinking regular water on day 14. Starting on day 16, 5-FU or vehicle (PBS) was injected intraperitoneally (ip) 4 times, once per week. After stopping tetracycline, the volume of subcutaneous tumors was reduced markedly. All of the treatments were statistically significantly different from control (mice fed tetracycline-water and treated with PBS), but the combination of induced IGF-Ir (tetracycline [−]) and 5-FU was the most effective in suppressing tumor growth. *P < compared with tetracycline (+) PBS. (C) Increased in vivo induction of apoptosis after truncated receptor derepression. The number of apoptotic cells by annexin V staining was counted in 20 (200×) fields per slide. Tumors from mice fed regular water and treated with either PBS or 5-FU showed more apoptotic cells than those fed tetracycline. *P < (D) RT-PCR showed in vivo expression of the truncated receptor in vivo after tetracycline withdrawal.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
IGF-Ir/tf expressing adenoviruses blocked IGF induction of Akt activity, induced cell death, and suppressed tumor growth in vivo. (A) Akt kinase assay showed that both truncated receptors blocked IGF-stimulated Akt kinase activity in HT29 cells and adenovirus-β-galactosidase failed to block this activity. The potency of Ad-IGF-Ir/950st in this assay for Akt was not different from that of Ad-IGF-Ir/482st. (B) Adenovirus IGF-Ir/950st decreased IGF-stimulated activation of Akt phosphorylation in 2 colon cancer cell lines (HT29 and HcT116) detected by Western blotting. (C) Both adenovirus IGF-Ir/tf constructs induced more cell death and enhanced 5-FU effectiveness in HT29 more than a control virus Akt-K179D, assayed by trypan blue staining. Adenovirus IGF-Ir/482st was the most effective among them. *P < (D) Subcutaneous HT29 tumors in nude mice were directly injected with 2 × 107 PFU of adenovirus on 5 successive days and the relative tumor volumes measured. Adenovirus IGF-Ir/482st was the most effective in inhibiting tumor growth. *P < ND, not done.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions