Hao MW, Liang YR, Liu YF, Liu L, Wu MY, Yang HX. Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines. World J Gastroenterol 2002; 8(2): 203-207
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World J Gastroenterol. Apr 15, 2002; 8(2): 203-207 Published online Apr 15, 2002. doi: 10.3748/wjg.v8.i2.203
Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines
Miao-Wang Hao, Ying-Rui Liang, Yan-Fang Liu, Li Liu, Ming-Yao Wu, Huan-Xing Yang
Miao-Wang Hao, Li Liu, Department of Internal Medicine, Tangdu Hospital, Xi'an 710038, Shaanxi Province, China
Ying-Rui Liang, Ming-Yao Wu, Huan-Xing Yang, Department of Pathology, Medical College of Shantou University, Shantou 515031, Guangdong Province, China
Yan-Fang Liu, Department of Pathology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Scientific Foundation of China, No. 39670298
Correspondence to: Dr. Miao-Wang Hao, Department of Internal Medicine, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Provioce, China. email@example.com
Received: September 14, 2001 Revised: October 1, 2001 Accepted: October 11, 2001 Published online: April 15, 2002
AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells.
METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only.
RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P = 0.000; expression level P = 0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P = 0.000; expression level P = 0.001). The growth rate of egr-1 -transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items.
CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.
Key Words: $[Keywords]
Citation: Hao MW, Liang YR, Liu YF, Liu L, Wu MY, Yang HX. Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines. World J Gastroenterol 2002; 8(2): 203-207
The tumor suppressorgene therapy research has become increasingly interested. Tumor suppressor genes have two categories: Type 1, they undergo mutation or deletion, such as P53, Rb, WT1 and BRCA1 etc. Type 2, they express little or nil and with rare mutation or deletion, such as cx34, maspin and integrin α6 etc. In designing the tumor gene therapy, the type II tumor suppressor genes are evidently more advantageous. We only need to induce or to promote more expression of the genes to get rid of many technical difficulties such as replacing the deleted gene or knocking out the mutated gene.
The early growth response gene, egr-1 (Zfp-6 in standardized genetic Nomenclature for mice, also known as NGFI-A, Krox-24, Zif-268, or TIS-8) is one of the important members of the immediate early gene family. It encodes a protein EGR-1 with 3 adjacent zinc-finger motifs, and structures that are present in many DNA-binding transcription factors. Its special zinc-finger structure can bind with “GC-rich” DNA regulatory elements and can control that particular gene to transcribe. The expression of egr-1 is closely related to cell growth and differentiation. Some researching results have revealed that EGR-1 can reverse the malignant phenotype of HT-1080 fibrosarcoma and ZR-75-1 mammary carcinoma cell lines, and can also EGR-1 inhibit the tumorigenicity and transforming growth of these cells[6-8]. egr-1 may act as a type II tumor suppressor gene.
Both hepatocellular carcinoma (HCC) and esophageal carcinoma (EC) are common malignant tumors in China. Many research works on HCC and EC[9-24], especially on the effects of the tumor suppressor genes such as P53, P16 and P21 etc[25-38] had been reported, but the reports about egr-1 for these two cancers are still lacking. In this paper, the transcription and expression of egr-1 in HCC, EC tissues and their normal counterparts were detected by in situ hybridization and immunohistochemistry in order to investigate the role of egr-1 in the cancerous process of HCC and EC. The constructed egr-1 eukaryo-expressing vector was transfected into HCC and EC lines, where there was absence of expression of egr-1, with a view to observe how the high level of exogenous expression of EGR-1 in the cancer cell influenced their growth, clone formation and tumorigenicity. The inhibitory action of egr-1 on HCC and EC is to be probed so as to provide an experimental evidence for the study of gene therapy of the two cancers.
MATERIALS AND METHODS
The plasmids, pAC-h-egr-1 (fragment of single and double enzyme digestion by BamH I and/or Sal I was determined by 10 g•L⁻¹ agarose gel electrophoresis. The size of the fragments was identical to that of plasmid map), containing the whole cDNA fragment of human wild-type egr-1 gene and pAC-Φ eukaryo-expressing vectors, were kindly granted by Dr. JG Monore, Pennsylvania University School of Medicine.
Human hepatocellular, esophageal carcinomas and breast tissue specimens were collected from the Pathology Department of Fourth Military Medical University and Medical College of Shantou University. All patients providing the specimens were not treated by radiotherapy or chemotherapy before operation. The specimens were fixed with 40 g•L⁻¹ formaldehyde solution (for in situ hybridization) and Carnoy solution (for immuno-histochemistry). Mouse brain tissues were from Balb/c mice.
The cell lines HHCC and SMMC7721 of HCC were purchased from Shanghai Cytology Institute, China. The EC cell line ECa109 was from Cytology Institute of Chinese Medical Academy. All the cell lines were cultured in RPMI 1640 medium supplemented with 100 mL•L⁻¹ new born bovine serum and grown in the circumstance of 37 °C, 50 mL•L⁻¹ CO2 and saturated humidity. Cell numbers were determined by Coulter counting method.
The LipofectamineTM, G418 and TRIzolTM total RNA extraction kit were purchased from Gibco/BRL Co. The WizardTM plus Minipreps DNA purification kit was the product of Promega Co. The polyclonal rabbit anti-human EGR-1 (C #-19) antibody was from Santa Cruz Co. In situ hybridization (POD) detection kit and SABC immunohistochemistry kit were from Boster Co, Wuhan, China. The AdvantageTM PCR purification kit was from Clontech Co, and γ-32P-dATP was provided by Yahui Biomedical Engineering Co, Beijing.
Probe preparation and labeling
The plasmid pAC-h-egr-1 was digested by both Sal I and BamH I, and the products were determined by agarose gel electrophoresis; the 400 bp DNA fragments were recovered by promega DNA purification kit. The recovered DNA was dissolved in 20 μL sterile three-distilled water and ultraviolet spectrophotometry was taken for quantitation analysis. γ-32P-dATP 5'end Label method was used for probe labeling. After alcohol precipitation, the probe was dissolved in TE; the specific radiation activity was determined by TAC method, and stored at -20 °C.
In situ hybridization and immunohistochemistry
In situ hybridization was performed according to the kit instructions on the formaldehyde-fixed and paraffin-embedded sections. Human normal breast and mouse brain tissue sections were used as positive controls. According to the SABC stain instructions, immunohistochemistry was done on the Carnoy-fixed or frozen section. Positive control was frozen section of human normal mammary adenosis, and negative control was primary antibody-blank.
Gene transfection and identification after transfection
Transfection was performed according to the legend of LipofectamineTM kit, and 48 h later selective culture was added with 600 mg•L⁻¹ G418.2 wk later, the cells and clones appeared all (the cells of blank control died). The concentration of G418 was changed to 400 mg•L⁻¹ G418 for maintenance. The clones were digested in situ, and transferred to a 6-well plate for amplification, which were used for detection of every item.
mRNA dot hybridization: 2 × 106 cells were collected and total RNA was extracted according to TRIzolTM total RNA extraction kit. The extracted RNA was dissolved in the RNase-free water. A absorbance value was determined, RNA quantity was calculated and purified was identified; stored at -80 °C; procedures of RNA dot blot was according to the directions.
Western blot hybridization and immunohistochemistry: Western blot hybridization was performed according to the directions. The “cell crawling slide” in logarithm stage was collected, rinsed with PBS × 2, fixed in 700 mL•L⁻¹ alcohol and stained by SABC kit. The positive control was frozen section of mammary adenosis, and the negative control was primary antibody-blank.
Assay of cell phenotype
FCM cell cycle examination: The transfected cells were digested by 2.5 g•L⁻¹ trypsin to a single cell suspension when they were 80% confluent, counted, 1 × 106 cells were fixed in 700 mL•L⁻¹ alcohol and FCM was performed.
Cell growth curve: cells of transfected and blank vector groups were digested by trypsin and counted in 1 × 103 cells. Each group was inoculated in 24-well plate, and 1 mL RPMI 1640 medium containing 100 mL•L⁻¹ FCS was added. In the culture period, cells of 3 wells were digested and counted every day, and the mean values were also calculated. Of the remainder, the medium was changed every 3-4 d and counted every 6-7 d according to the cell growth conditions, growth curves were then made.
Examination of plate cloning: HCC and EC lines, which had been transfected with egr-1 gene, G418 selected and amplified were digested to a single cell suspension. Cells counted were inoculated into 6-well tissue culture plates; each well contained 100 cells. The control cells were transfected with blank vector. Changing the medium 1-2 times per wk; by about 2 wk plates were taken out to observe the clone formation; fixed in methyl alcohol, Giemsa stained, clones over 50 cells were counted, and the mean value was adopted and clone formation rates were made.
Nude mice tumorigenicity: After trypsin digestion, the cells of the experimental and control groups were inoculated into nude mice subcutaneously with 1 × 107 cell (about 0.1-0.2 mL) for each mouse. About 3 wk later the mice were killed according to the tumor growth, and then pictures were taken. The tumor mass was separated carefully and weighed, and the daily growing mass was calculated then. Tumor tissues were fixed, paraffin embedded, HE stained and observed microscopically.
Results were expressed as mean ± SE. To compare the mean values, Stata statistical software was used. P < 0.05 was considered significant.
Egr-1 mRNA and protein expression in HCC and EC
In situ hybridization and SABC immunohistochemistry staining for egr-1 mRNA and protein in human HCC, EC tissues and their normal counterparts were performed. Little or no egr-1 transcription was detected in HCC and normal liver tissues. Higher transcription of egr-1 was detected in hepatocellular paracancerous tissues such as large cell hyperplasia (LCD) and “atrophic-like liver plate (reluctant LCD)”. egr-1 mRNA signal was present in the cytoplasm. EGR-1 expression in HCC, normal liver and hepatocellullar paracancerous tissues was consistent with the transcription (When EGR-1expression in HCC compared with the expression in the paracancerous tissue, P = 0.143. Table 1, Figure 1). EGR-1 protein signal was in the nucleus. Little or no egr-1 transcription was detected in the aggressive EC and normal esophageal epithelial tissues (not including basal layer cells). Higher transcription of egr-1 was detected in dysplastic esophageal epithelium. EGR-1 expression in aggressive EC, normal esophageal epithelium and dysplastic tissues was also consistent with the transcription (Table 2, Figure 2).
Table 1egr-1 transcription and expression in HCC tissue and its normal counterparts.
Figure 2 Positive signal of EGR-1 mRNA in the cytoplasm of moderately dysplastic esophageal epithelium.
SP × 400
Egr-1 mRNA and protein detection in transfected tumor cells
mRNA level (mRNA dot blot): The total RNA of egr-1 and vector transfected HCC and EC cells were dot hybridized by 32P labeled egr-1 probe. The result revealed that egr-1 group had stronger signals than that of the control.
Western blot and immunohistochemistry: The proteins of egr-1 and vector transfected HCC and EC cells were extracted and quantitatively analyzed. Then both groups with identical quantitative proteins were determined by SDS-PAGE; electrical transfer onto the nitro-cellulose membrane was made, and Western blot hybridization was performed. The results revealed that in egr-1 transfected groups of both HCC and EC cells, there were strongly stained bands just at the molecular mass of 59 ku, which was identical to that of EGR-1, whereas in all the controls there were no such bands present. This suggested that the tumor cells transfected by egr-1 could strongly express EGR-1 protein. The results of immunohistochemistry by primary antibody EGR-1 and SABC method showed 50%-70% positive staining of egr-1 transfected cells were, while in the controls the results were all negative; which was consistent with that of the Western blot.
Malignant phenotype examination
Curves of cell growth The growth rates of the egr-1 transfected HHCC liver carcinoma cells and EC109 esophageal carcinoma cells were obviously slower than that of the controls. But the difference was not significant in the SMMC7721 liver carcinoma cell line (Figure 3).
Figure 3 Cell growth curve of liver and esophageal carcinoma cells transfected egr-1 and vector
FCM cell cycle assay In egr-1 transfected HHCC, the number of fraction of S phase accounted for 0.15, lower than that of the controls (0.28, P = 0.038); i.e., the proportion of S phase was 0.46 lower than that of the control (Table 3). In ECa109, the number of fraction of S phase accounted for 0.18 (control 0.27, P = 0.048), and was 0.34 lower than that of the control. In SMMC7721 (HCC), there was no marked difference between the experimental and the control groups (0.21 to 0.22, P = 1.000). As for the G1 and G2/M phases, there were neither significant differences between experiment groups and control groups.
Table 3 Cell cycle of HCC and EC cells expressing EGR-1 with FCM (number of fraction).
In HHCC cell line, the clone forming rate of transfected cells was 49.0% ± 10.6%, 42.7% lower than that of control 91.7% ± 6.1%. In ECa109 line, the clone forming rate of transfected cells was 45.0% ± 5.3%, 32.3% lower than that of control 77.3% ± 3.1%. In SMMC7721, There were no statistical difference between the experimental group 26.0% ± 2.6% and the control group 23.3% ± 2.1% (Figure 4).
Figure 4 Clone formation rates of exogenous high EGR-1 expression HCC and EC
Nude mice tumorigenicity
All the inoculated nude mice had tumor mass growth except in one case (one of the three SMMC7721/φ inoculated mice). In HHCC cell line, the tumor growth rate of transfected group was 80.7%, lower than that of the control (P = 0.001). In ECa109 cell, the growth rate of transfected group was 72.6%, also lower than that of the control (P = 0.001). In SMMC7721, there was no growth rate difference between the experimental and the control group (P = 0.681) (Table 4).
Table 4 Nude mice tumorigenicity of gene transfected groups in HCC and EC cells.
Althrough egr-1 is a member of immediate early genes, its expression is also elevated during the process of growth and differentiation. Observations suggest that EGR-1 have pleiotropic roles such as growth, development, differentiation etc. This is Supported by the finding that the GC-rich DNA-binding element for egr-1 (GCE) is present in a large number of gene regulating regions, including growth factors, signal transduction genes, other transcription factors and oncogenes. In this report, we showed that egr-1 transcription and expression decreased obviously in HCC and EC tissues compared with their paracancerous tissues. Another (large) study also indicated that human small cell lung tumors express little or no egr-1 mRNA compared with adjacent normal tissues, further supporting our findings. Except that the egr-1 gene was deleted only in all 5q- syndrome cases, egr-1 expression was profoundly decreased in a number of other human tumor cell lines such fibroblastoma, glioblastoma, osteosarcoma and lung cancer.Therefore it seems likely that the inactivation of egr-1 expression is a generalized phenomenon in the developmental process of a number of tumors.
Egr-1 is ubiquitously expressed at low levels but accumulates to relatively high levels in only a few adult organs as brain, heart, lung, kidney and breast. We showed here that normal liver and esophageal epithelial tissues expressed undetectable level of egr-1, but the egr-1 gene was activated in the tissues of hepatic paracancerous tissue such as LCD and “atrophic liver plate” (reluctant LCD)and dysplastic tissue of esophageal carcinoma. During regeneration of normal liver tissue, increased egr-1 expression is induced. In mouse fibroblast NIH3T3 cells, egr-1 expression increases two-fold 10 min after UV irradiation, which rises to a maximum (eightfold induction) after 2 h. Human HT1080 fibrosarcoma subclone, cells H4, express little or no egr-1. Phorbol ester treatment only can elicit a small increase in egr-1 expression in H4, in contrast to the normally rapid, high transient expression of egr-1 observed after the addition of a wide range of stimulating agents to normal or immortalized cell lines. Therefore, the meaning of little or no egr-1 expression in liver and esophageal carcinomas is different to their counterparts. egr-1 gene in the hepatic paracancerous tissue such as LCD and dysplasia of esophageal carcinoma can be activated when they are stimulated, but the stimulations is invalidated in carcinomas. The decreased expression of egr-1 may play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. To confirm the development of liver and esophageal carcinomas involves the inactivation of egr-1. It would deserve further investigation of the effects of exogenous EGR-1 on the growth and malignant phenotypes of the cancer. Like tumor suppressor gene WT1, EGR-1 can also bind with GCE elements, this suggests that EGR-1 might have inhibitory activity on cancer growth. In the experimental report, EGR-1 could reverse the malignant phenotype of v-sis transformed NIH3T3 cells. The results of this paper also showd that stably expressed EGR-1 could inhibit the cancer's growth rate, reduce the number of fraction of S phase, decrease the plate clone formation rate and reduce the tumorigencity of HHCC and ECa109 cell lines. In recent years, studies have revealed that in human tumors, EGR-1 expression suppressing tumor cell growth is a common phenomenon. In human HT-1080 fibrosarcoma cells, the growth rates in 6 groups of expressing different EGR-1 level are inhibited proportionately and are dose-dependent. The total inhibition rate of these cells' tumorigencity is over 50%. The growth of human ZR-75-1 breast carcinoma, U251 glioblastoma and SAOS-2 osteosarcoma cells has also been demonstrated to be suppressed by EGR-1 in different degree. However, in this experiment EGR-1 did not suppress the malignant phenotypes of SMMC7721, HCC cell line, this might be due to different transformation mechanism in this cell line, which needs investigation further.
The mechanism of EGR-1 inhibition on cancer growth is complicated, which interacts with TGF-β1, PDGF and WT1. TGF-β1 specifically inhibits PDGFβ-dependent cells' growth, while EGR-1 inhibits the malignant phenotype and growth of the PDGFβ/v-sis that were transformed from NIH3T3 cells. This suggests that EGR-1 is closely related with TGF-β1 and PDGF. Meanwhile, EGR-1 can also activate the transcription of TGF-β1 in a dose-dependent manner, this activation might be one of the potential mechanisms of EGR-1 of inhibiting tumor growth. Interestingly, WT1 can bind with the GC element of TGF-β1 promoter, and then inhibit the activity of that promoter (but this reaction is weaker than that of inhibition on PDGFα). One recent study has shown that the inhibitory reaction of EGR-1 on cancer growth is also by inducing the expression of TGF-β1, fibronectin, P21 and focal adhesion kinase (FAK) in human fibrosarcoma cell line. EGR-1 may also regulate cell interaction with the extracellular matrix by synergistic induction of TGF-beta1, FN, and PAI-1 in human HT-1080 glioblastoma cells and directly transactivates the fibronectin gene and enhances attachment of human glioblastoma cell line U251. Other reports show that the suppression by egr-1 also involves down-regulation of bcl-2 and stimulates apoptosis by transactivation of the P53 gene.
Recent studies[6,8,50] as well as this experiment suggest that EGR-1 can act as a tumor suppressor factor. EGR-1 has been identified to be little or no expression in several human malignant tumors such as fibroblastoma, glioblastoma, osteosarcoma, lung cancer, breast carcinoma (including cancer cells and tumor tissues)[1,6], as well as hepatocellular carcinoma and esophageal carcinoma in this experiment. Another (larger) study also indicates that human small cell lung tumors express little or no egr-1 mRNA compared with adjacent normal tissues. Furthermore, 5q-syndrome is the exclusive tumor with egr-1 gene deletion. Hence we suspect that egr-1 might be belonged to typeIItumor suppressor genes as mentioned above. The reintroduction of egr-1 gene products by drugs might be a promising approach to normalize growth regulation, which can avoid the difficult problem of replacing defective gene DNA.
Thanks to Prof. Bo-Rong Pan in the Oncology Center of Xijing Hospital for his help in the preparation of this article.
Edited by Wu XN
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