Jen-Fu Chiu

Tamoxifen induces TGF‐β1 activity and apoptosis of human MCF‐7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF‐7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF‐7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM‐2 and TGF‐β1 mRNAs in MCF‐7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen‐induced pS2 gene was strongly suppressed. The biological activity of TGF‐β was increased at least fourfold in the media from MCF‐7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF‐7 cells and it may be mediated by the secretion of active TGF‐β.

Down-regulation of Manganese-Superoxide Dismutase through Phosphorylation of FOXO3a by Akt in Explanted Vascular Smooth Muscle Cells from Old Rats

Manganese-superoxide dismutase (MnSOD) is one of the major cellular antioxidant defense systems. To study the effect of age on the regulation of MnSOD in the vasculature, we compared MnSOD expression and its transcriptional regulation in explanted vascular smooth muscle cells (VSMC) isolated from old (24 months old) versus young (6 months old) rats and grown in a normal (5 mm) or high (12.5 and 25 mm) glucose or tumor necrosis factor α (5 ng/ml) environment to induce oxidative stress. Both MnSOD protein and activity were reduced in VSMC from old compared with young animals. FOXO3a, a member of the family of Forkhead transcription factors, interacted with the promoter of the rat MnSOD gene at a specific binding site. Inhibition of FOXO3a transcription with small interfering RNA led to a reduction in MnSOD gene expression. VSMC from old rats had increased phosphorylated FOXO3a at Ser253, which paralleled the reduction of MnSOD protein. Treatment of VSMC with 5 nm insulin-like growth factor-1 induced phosphorylation of Akt and FOXO3a over time, repressing FOXO3a DNA binding and consequently MnSOD gene expression. Furthermore, Akt activity was selectively increased in VSMC from the old, supporting the hypothesis that increased age-related Akt activity might be responsible for the phosphorylation and inactivation of FOXO3a, which in turn down-regulates MnSOD transcription.

Arsenic induces oxidative stress and activates stress gene expressions in cultured lung epithelial cells.

Chronic exposure to low levels of arsenic can cause lung cancer. However, the cellular and molecular mechanisms for lung cell transformation in response to arsenic are not known. These studies investigated the hypothesis that low levels of arsenic increase intracellular oxidant levels, promote production of mitogenic transcription factors and antioxidant enzymes. Initially, arsenic decreased GSH cellular level and rapidly increased to 280% of GSH level in nonexposed lung cells in 24 h. Buthionine sulfoximine (BSO) potentiated the arsenic toxicity of lung epithelial cells (LEC). Exposure of LEC to 5 μM arsenite cause time‐dependent increase in γ‐glutamylcysteine synthetase (γ‐GCS) expression. Our data demonstrated that arsenic induced the heavy subunit of γ‐GCS (γ‐GCS‐HS) mRNA levels as early as 4 h as compared to the control level. It significantly increased (sixfolds) γ‐GCS‐HS mRNA expression after 8 h of treatment. The activation of AP‐1 transcription factors may also play a regulatory role in this process. Significant elevations in c‐fos and c‐jun mRNA levels were observed within 30 min after exposure to arsenic and by enhancement of AP‐1 DNA binding activity and transactivation activity. Responsiveness of LEC to oxidative stress caused by arsenic exposure was further evaluated with mobility shift assay involving redox‐sensitive transcription factor NF‐κB. The specificity of binding was verified by an antibody‐supershift. The NF‐κB DNA binding activities increased more than twofold 30 min after exposure to arsenic and returned to control levels after 4 h of treatment. It remains to be determined whether NF‐κB plays a role in the As‐induced apoptosis or alternatively in attempting to protect the cells from As‐induced cell death by upregulating the expression of resistance factors. J. Cell. Biochem. 87: 29–38, 2002.

The expression of oncogenes in human developing liver and hepatomas

Oncogene expression was examined in the human fetal liver and human hepatomas. Erb (B), erb (A+B), Ha-ras, myc, fos and fms oncogene expression elevated in certain stages of fetal liver development and in hepatoma as compared to the normal adult human liver. In contrast, rel, src, mos, sis, myb, Ki-ras and bas oncogenes showed no apparent change of their mRNA levels during fetal liver development and in hepatoma. Further study of erb B oncogene expression in human cirrhotic liver and hepatoma demonstrated a strong correlation between erb B expression and alteration of its gene structure.

Dexamethasone inhibits α-fetoprotein gene expression in developing mouse liver

Abstract The effect of dexamethasone on α-fetoprotein gene expression was studied in newborn mice. The treatment of 3 day old mice with 1 μg/gm body weight of dexamethasone twice a day reduced the α-fetoprotein level in serum to about 2.5% of the normal control level after 4 days of administration. The level of α-fetoprotein mRNA sequences in the cytoplasm decreased to about 1% of the control over this same period. Furthermore, only an extremely low concentration of α-fetoprotein mRNA sequences in nuclear RNA could be detected even at a high Crot. These results suggest that dexamethasone inhibits transcription of the α-fetoprotein gene in newborn mice.

Hepatitis B virus DNA integration and expression of an erb B-like gene in human hepatocellular carcinoma

Southern blot studies on Hepatitis B Virus (HBV) DNA integration in 13 human hepatocellular carcinomas (HCCs) patients revealed the presence of several distinct HBV integration sites in different human liver disease patients. In one HCC patient the DNA fragment containing the HBV integration also hybridized to an erb B probe. The erb B/HBV co-migrating DNA fragment was cloned and sequenced, and showed that HBV DNA is integrated next to a cellular DNA fragment which is homologous to the tyrosine protein kinase domain of the human epidermal growth factor receptor gene and other cell surface receptor genes. The virus-integrated cellular DNA sequence is expressed in this HCC patient, suggesting a possible role for this gene in hepatocarcinogenesis.

Differential expression of cellular oncogenes during rat liver development

The expression of a number of proto-oncogenes (myc, erb B, Ha-ras, bas, rel, mos, sis, myb, ki-ras, fms, src and fos) was studied in developing rat liver. Northern blot hybridization shows that cellular counterpart of erb B, Ha-ras, and fos oncogenes were in an early stage of liver development, and the expressions of these proto-oncogenes gradually decreased as the liver developed, while c-myc transcript was found only in the rat fetal liver. The transcripts of these oncogenes were found in high level in Morris hepatoma 7777. Bas proto-oncogene was found in high expression at early stages of rat liver development but was not in hepatoma 7777. The expression of other proto-oncogenes studied (src, fm, rel, mos, sis, myb and ki-ras) did not change significantly during liver development and was almost the same in hepatoma and normal adult liver. Southern blot analysis demonstrates that gene amplification and apparent gene rearrangement were not responsible for the change in expression of erb B, Ha-ras, myc and fos proto-oncogenes. Our study gives further evidence that erb B, myc, Ha-ras and fos proto-oncogenes are involved in the control of cell growth and in the process of rat hepatocarcinogenesis.

Induction of F9 cell differentiation by transient exposure to retinoic acid

The F9 cell is a mouse embryonal teratocarcinoma which can be induced to differentiate into visceral endoderm by treatment with retinoic acid (RA). Treatment with RA in conventional studies was carried out in the constant presence of RA. Here we demonstrate that treatment with RA can be as short as 3 hrs to induce differentiation of F9 cells. Morphology, alpha-fetoprotein gene activity, and temporal patterns of F9 cell differentiation are the same with both short- and long-term treatment with RA.

Antigenic changes in nuclear chromatin in 1,2-dimethylhydrazine-induced colon carcinogenesis

Chromosomal nonhistone protein‐DNA complexes prepared from rat colon adenocarcinoma were used to produce tumor specific antisera in rabbits. The antisera reacted specifically in complement fixation tests with chromatins isolated from rat colon tumor and not with those from a number of other rodent tumors and normal tissues, including normal rat colon epithelial cells. Tumor specificity of this antisera was also demonstrated by their cross reactivity with human and mouse colon adenocarcinomas. Immunohistochemical localization of the antigens by the horseradish peroxidase bridge technique demonstrated their presence in the nuclei. The changes in the immunospecificity of the nuclear antigens in chromatin were studied in colon of rats treated with 1,2‐dimethylhydrazine. A significant change in the immunospecificity of the chromatin occurred as early as the fourth week after 1,2‐dimethylhydrazine treatment was begun.

Comparison and evaluation of gene therapy and epigenetic approaches for wound healing

During the past decade considerable evidence has mounted concerning the importance of growth factors in the wound healing process both for cell replication and for stimulating reparative cells to synthesize and secrete extracellular matrix components. During normal wound healing the growth factor concentration has to be maintained at a certain level. If the growth factor concentration is too low, normal healing fails to occur. Whereas if the growth factor concentration is too high due to either over‐expression of the growth factor or too much growth factor being applied to the wound, aberrant wound healing will occur. One approach for controlling the amount of growth factor at the wound site during normal healing is through gene therapy and the titration of gene dosage. However if a narrow window exists between the beneficial therapeutic effect and toxic effects with increasing gene dosage, an agent may be necessary to give in combination with gene therapy to regulate the over‐expression of growth factor. In addition to genetic approaches to regulate wound healing, epigenetic approaches also exist. Antisense oligodeoxynucleotides have been shown to regulate wound repair in certain model systems and to determine the protein(s) necessary for normal wound healing. A novel approach to regulate the activity of collagen genes, thereby affecting fibrosis, is to use a sense oligodeoxynucleotide having the same sequence of the cis element which regulates the promoter activity of a particular collagen gene. This exogenous oligodeoxynucleotide will compete with the cis element in the collagen gene for the trans‐acting factor which regulates promoter activity. These epigenetic approaches afford the opportunity to regulate over‐expression of growth factor and therefore preclude the potential toxic effects of gene therapy. Both genetic and epigenetic approaches for regulating the wound healing process, either normal or aberrant wound healing, have certain advantages and disadvantages which are discussed in the present article.